PROJECT SUMMARY/ABSTRACT Hypervirulent, drug-resistant strains of Clostridium difficile now greatly contribute to the overall morbidity and mortality of C. difficile infection (CDI), resulting in relapse rates of up to 35%, an estimated 29,000 deaths per year, and a national burden in excess of $4.5 billion annually. There is an urgent need for the characterization of novel antibacterial targets for the treatment of CDI. One novel and potentially selective target is the bacterial enzyme FabK, enoyl-acyl carrier protein (ACP) reductase II, a key enzyme in the bacterial fatty acid synthesis pathway (FAS II). Inhibition of enzymes in the FAS II pathway has been shown to result in a strong antibacterial effect, as with the FabI (enoyl-ACP reductase I) inhibitors triclosan, isoniazid, and other FabI inhibitors currently in clinical trials. C. difficile expresses FabK as its sole enoyl reductase, an isozyme that is structurally and mechanistically distinct from FabI. We hypothesize that inhibitors of the CdFabK enzyme will show selective activity against C. difficile, while causing minimal disruption to the normal flora of the lower bowel. This hypothesis was formulated based upon existing literature demonstrating a species-specific distribution of enoyl-reductase isozymes in gut flora, known inhibitors with selective activity against the FabK enzyme, and our own preliminary data demonstrating target essentiality. Here, our objectives are to validate the FabK enzyme in C. difficile as a druggable target for narrow-spectrum CDI therapy. To achieve these objectives, we have organized a skilled team of scientists with expertise in C. difficile microbiology, protein biochemistry, structural biology, synthetic and computational chemistry, who will pursue the following specific aims: 1. Microbiological and in vivo validation of FabK as a narrow- spectrum target in C. difficile. Our working hypothesis for this aim is that C. difficile cannot overcome FabK/FAS-II inhibition by pathway bypass in the presence of exogenous fatty acids. We will utilize a series of in vitro and in vivo studies using inducible FabK over- and under-expressing C. difficile strains and tests on key gut bacteria to address this hypothesis. 2. Structural and chemical validation of FabK as a druggable target in C. difficile. The working hypothesis for this aim is that the FabK enzyme can be inhibited by small- molecule compounds resulting in anti-difficile activity. We will test this hypothesis by utilizing a combined approach of synthetic optimization of known FabK inhibitors, coupled with synergistic virtual and experimental screening, to develop and optimize inhibitors with both high affinity and selectivity for CdFabK. The studies proposed here will advance our biological understanding of the gut flora FAS-II requirements, validate an attractive antibacterial drug target, shed new light on fatty acid regulation in C. difficile and provide strong proof of principle for further development of CdFabK inhibitors as a new therapeutic strategy for treatment of drug- resistant CDI.